A post-transplant infection by Nocardia cyriacigeorgica

Nocardia are Gram-positive, acid-fast, filamentous bacteria that cause opportunistic infections in susceptible populations. We describe a case of post-transplant infection of pulmonary nocardiosis caused by the rare strain Nocardia cyriacigeorgica and the challenges faced in reaching a definitive diagnosis. This case report emphasizes on keeping nocardiosis as a differential diagnosis in transplant recipients, as this disease is largely underdiagnosed and underreported.


INTRODUCTION
In patients receiving immunosuppressive therapy after renal transplantation, the possibility of opportunistic infections increases several fold.One such life-threatening opportunistic infection is nocardiosis [1], caused by organisms that are mainly inhabitants of water and soil [2].Among the various species causing the said disease, Nocardia asteroides complex is most common, followed by Nocardia brasiliensis and Nocardia otitidiscaviarum [3,4].
Looking at the spectrum of diseases caused by Nocardia, it most often involves the respiratory system, but it also shows affinity towards the brain, skin and soft tissues [5].Although it rarely affects immunocompetent patients, a positive case is most likely a result of inoculation of the organism directly through foreign object penetration into the body [4].Incidence of nocardiosis after solid organ transplant is rare in comparison to other opportunistic infections, amounting to only ~0.6 % [6].T-cell depleting therapies, including antithymocyte globulin, increase the risk for opportunistic infection.Infected patients mostly present with signs and symptoms of lower respiratory tract infections that do not respond to routine empirical antibiotics, along with progressive deterioration in the general condition of the patient [2].Microbiological diagnosis of such cases can be tricky given the paucity of organisms in respiratory samples after ruling out other common causes of the infections such as tuberculosis, bacterial pneumonia and invasive fungal disease.Traditional techniques along with recent advancements in microbiology can be combined to reach an accurate diagnosis in such cases [7].Here, we highlight a case of pulmonary nocardiosis in an immunocompromised patient post-renal transplantation caused by a rare species, Nocardia cyriacigeorgica.

CASE PRESENTATION
A 41-year-old male with a history of grade 5 chronic kidney disease, diagnosed 2 years ago, had an HLA 6/6 mismatch livingrelated renal transplant: wife to husband in the month of April 2022.Post-transplant, the patient was put on immunosuppressive medications.Approximately 2 months after the transplant, the patient gradually developed fever, which was intermittent in nature, and a cough with expectoration, which exacerbated on supine position.To evaluate the aetiology, sputum and bronchoalveolar lavage (BAL) sample collection was done, and the specimens were sent for microbiological investigation.Chest X-ray, ultrasonography of the thorax and high-resolution computed tomography (HRCT) revealed bilateral nodular consolidations that were progressive in nature (Figs 1 and 2).The patient's sputum and BAL culture was negative for bacteria, fungi and Mycobacterium tuberculosis.As a last resort, a closed transbronchial lung biopsy was done given the insignificant sputum and BAL studies, and progressive deterioration in the condition of the patient.A Gram-stain smear of excised tissue exhibited a Gram-positive branching filamentous structure, and Ziehl-Neelsen (ZN) stain of the same revealed a weak acid-fast branching filamentous structure.A modified acid-fast staining using 0.5-1 % sulphuric acid as a decolourizer (rather than the conventional 20 % sulphuric acid) serves as an extremely useful guide for the diagnosis of Nocardia, aligning with the observations of Gramstain and ZN stain.The modified ZN revealed acid-fast branching filamentous bacilli.The inoculation of the tissue sample was  done on blood, chocolate and MacConkey's agar.On blood and chocolate agar, dry, chalky white, medium-sized colonies were observed, whereas no colonies were observed on MacConkey's agar.To observe the gross appearance in liquid media, tissue was also inoculated into Robertson's cooked meat (RCM) broth, in which large, chalky white pellicles were found.Smears from the colonies of both blood and chocolate agar were subjected to Gram, ZN and 1 % modified ZN staining, and subsequent microscopy revealed a Gram-positive, acid-fast, branching, filamentous bacilli.The colonies were also inoculated onto Mueller-Hinton agar by lawn technique to check for drug susceptibility, as a method to determine the possible group of Nocardia causing the condition (Fig. 3).Drugs used for differentiation by susceptibility were tobramycin, amikacin, gentamicin and erythromycin [7].The cultured isolate was found to be susceptible to tobramycin, amikacin and gentamicin, but was resistant to erythromycin  (Fig. 4).Based on this pattern of susceptibility, it was suspected that the organism was among the following, i.e.N. asteroides I/ VI, N. brasiliensis, Nocardia pseudobrasiliensis and N. otitidiscaviarum.For complete identification of the species, a MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) MS ID test (MALDI Biotyper Sirius; Bruker) was performed on the sample.The isolate was identified as N. cyriacigeorgica.The patient was started on injection meropenem 1 g, twice daily for 10 days, and oral co-trimoxazole once daily (sulfamethoxazole 800 mg/trimethoprim 160 mg) for 25 days, and further was put on prophylaxis with oral co-trimoxazole -200 μg for 3 weeks.The patient's response to treatment was favourable.Follow-up for the patient was uneventful and subsequently a lung biopsy was done for confirmation (as the patient was immunocompromised and to avoid further complications), which revealed no similar bacterial culture.

DISCUSSION
Nocardia species are known to be Gram-positive, filamentous, branching bacilli that are also partially acid-fast.The prevalence of nocardiosis is rare in comparison to other bacterial infections; however, under certain conditions, it should be kept as a differential diagnosis that should not be ignored [4].The incidence of Nocardia infections after a solid organ transplant can be between 0.04 and 3.5 % [8], and the incidence in renal transplant patients can be between 0.04 and 1.2 % [9].According to a case-control study conducted by Peleg et al., the risk factors for nocardiosis in solid organ transplant recipients can be attributed to high-dose steroid therapy, infection with cytomegalovirus (CMV) and high median calcineurin inhibitor levels in the preceding 30 days (>300 ng ml −1 for cyclosporine and >15 μg ml −1 for tacrolimus) [6].It was also noted that patients receiving tacrolimus were at a higher risk of contracting nocardiosis than those receiving cyclosporine [10].Our patient also had tacrolimus trough levels of 6 μg ml −1 and high-dose corticosteroids to avoid organ rejection due to HLA mismatch.It is reported that Nocardia infections usually present within a period of 6 months and rarely 1 year post-transplant, similarly our patient presented with the typical symptoms of cough, fever and breathlessness [3].Due to the lack of distinctive clinical findings or radiological evidence indicative of a Nocardia infection, Gram-staining of sputum samples remains a vital tool for diagnosis [3,5,7].Bilgarnia et al. and Flohr et al. reported two other cases of pulmonary nocardiosis.Both cases underwent re-transplantation, and preconditioning was done using plasmapheresis and rituximab.Post-transplant, these patients were maintained on mycophenolate mofetil, tacrolimus and steroids [11,12].We received multiple sputum specimens, but all their Gram-stain reports were insignificant and ZN staining was negative for the presence of acid-fast bacilli.For better diagnostic sensitivity, BAL samples were then taken, which proved to be a failure.As prior investigations proved to be inconclusive, more invasive techniques were indicated [13].Invasive samples like BAL were sent, which were negative for infections like mycobacterium, but because of the persistent radiological evidence, a lung biopsy was done that proved Nocardia species, an opportunistic infection.The lung biopsy samples assisted in isolating the causative organism by providing the above-mentioned picture in staining and culture.Antibiotic susceptibility for Nocardia can be identified by broth microdilution [14], or by the Kirby-Bauer disc diffusion method [15] (Table 1).In our study, broth microdilution was not done due to technical reasons.An accurate diagnosis was made because of the cultures; after which, treatment was initiated promptly to deal with infection.MALDI-TOF MS test, wherever applicable, can be used to diagnose such cases, as it gives fast and reliable results for such difficult-to-diagnose pathogens [16].Prophylactic treatment for Nocardia usually involves long-term co-trimoxazole.Alternatively, drugs such as amikacin, imipenem, meropenem and third-generation cephalosporins have been shown to have efficacy against Nocardia in vitro [17].Line 234: correction has been made in the manuscript as requested by reviewer 1.

End of changes requested by Reviewer 1
Reviewer 2: Line 26: The bacteria in the genus Nocardia are filamentous.It recommends using the appropriate word because they are not true bacilli.
Line 26: As suggested by reviewer 2, we have changed the terminology used in Line 26.We have now referred nocardia as gram positive, acid fast filamentous structures.
Line 96: Instead of " A 1% modified ZN… ", show how to do modified acid-fast staining correctly.Take into account that most Nocardia species are partially acid-fast when using modified acid-fast staining like Kinyoun method.
Line 99/100: As requested by reviewer 2, the change has been implemented in Line 99/100..

-The history of medical interventions, including each immunomodulation, medicine, and any other patient's risk factors, should be provided in the case presentation section.
Line 88: As requested by reviewer 2, changes have been made in Line 88.
Patient's immunomodulation has been discussed in the "discussion section" of the manuscript.
-Since the isolate has already been identified using the accurate MALDI-TOF method, it is not required to describe a nonspecific identification method like the susceptibility pattern to four antibiotics.
In order to come to a stepwise, systematic diagnosis for Nocardia, we have described our thinking process.As we wanted to narrow down the species, the susceptibility pattern to antibiotics was used.

End of changes requested by Reviewer 2
Reviewer 3: 1. Description of the case(s): Good description of case but can be done better by including some diagnostic parameters that support the evidence of infection by an opportunistic microorganism.
The patient was immunocompromised following a renal transplantation, and presented with symptoms of an infection.Patient was having a persistent cough, and radiological investigation was revealing a consolidation (supporting evidence of this has been attached in FIGURE 1 and 2).After ruling out the endemic causes of such a presentation (TB), a lung biopsy was done, and an opportunistic infection was diagnosed.

Presentation of results: The results can be presented in a better fashion by presenting the diagnostic parameters along the course of hospital stay, showing improvement on diagnosis by department of microbiology and how the antibiotic susceptibility helped the treatment of the individual.
The authors feel that the results are being presented in best possible way, as per our resources and expertise.We have made some overall changes in the manuscript to depict the change in patients condition.Invasive samples like BAL were sent which were negative for infections like mycobacterium, but because of the radiological evidence, a lung biopsy was done, which proved Nocardia species, an opportunistic infection.An accurate diagnosis was made because of the cultures, after which, treatment was initiated promptly to deal with nocardia infection.The patient subsequently improved on co-trimoxazole.

How the style and organization of the paper communicates and represents key findings. The organization of the paper is good and represents most of the key features.
No changes 4. Literature analysis or discussion: Not up to the mark.Please discuss more cases with their antibiotic susceptibility patterns and please provide a substantial reason as to why broth micro dilution was not performed for the isolate.
Line 153/154: Broth microdilution was not done due to technical reasons.

Any other relevant comments: please improve the discussion section as very few studies discussing the antibiotic susceptibility and other cases of infections with the same isolate. Please provide reason for not performing broth micro dilution
Multiple changes to the discussion section has been made.Broth microdilution was not done due to technical reasons.A table mentioning the methods used for antibiotic sensitivity testing for nocardia species by Kirby bauer and broth microdilution has been added.

End of changes requested by Reviewer 3
Comments: 1. Description of the case(s): Good description of case but can be done better by including some diagnostic parameters that support the evidence of infection by an opportunistic microorganism.2. Presentation of results: The results can be presented in a better fashion by presenting the diagnostic parameters along the course of hospital stay, showing improvement on diagnosis by department of microbiology and how the antibiotic susceptibility helped the treatment of the individual.3. How the style and organization of the paper communicates and represents key findings.The organization of the paper is good and represents most of the key features.4. Literature analysis or discussion: Not up to the mark.Please discuss more cases with their antibiotic susceptibility patterns and please provide a substantial reason as to why broth micro dilution was not performed for the isolate.-Since the isolate has already been identified using the accurate MALDI-TOF method, it is not required to describe a non-specific identification method like the susceptibility pattern to four antibiotics.

Please rate the quality of the presentation and structure of the manuscript Satisfactory
To what extent are the conclusions supported by the data?Strongly support

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes Reviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000569.v1.4 © 2023 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.

Anonymous.
Date report received: 17 March 2023 Recommendation: Minor Amendment Comments: Overall well written manuscript.These rare infections are important in patients with risk factors and timely diagnosis and treatment is necessary for good outcomes.Few minor suggestions to improve the manuscript 1.
C a s e report a. Developed fever instead of 'a fever' b.Exacerbated on supine position instead of supination c.Can remove -As a part of evaluation CXR, USG and CT were done.Instead, can write CXR and USG findings.
d. Can mention what all sputum and BAL investigations were done and negative (bacterial culture, fungal culture, TB, others) e.How was lung biopsy done ?Open lung biopsy or other method?f.Why was a repeat lung biopsy was done when patient was clinically improving ?g.Was the patient put on prophylaxis for nocardiosis after clinical recovery/cure? 2. Discussion a. Tacrolimus levels -units in ng/mL 3. Figure 1 -Legend A encircles the largest nodule -it should be consolidation Please rate the quality of the presentation and structure of the manuscript Good

Fig. 1 .
Fig. 1.Coronal section of computed tomography scan showing multiple nodules and mass-like consolidations in the left lung.A encircles the largest consolidation.

Fig. 2 .
Fig. 2. Axial section of computed tomography scan.B encircles the largest consolidation in the left lung.

Fig. 3 .
Fig. 3. Growth of colonies of the isolate on Mueller-Hinton agar.

Table 1 .
Various case reports dealing with Nocardia species isolation and their findings Nocardia should be kept as a differential diagnosis in renal transplant recipients presenting with a pulmonary infection.Invasive diagnostics can help the patient out with an early diagnosis of nocardiosis and prevent disease dissemination.If needed, the use of advanced techniques such as MALDI-TOF MS should be sought for an early and reliable diagnosis.

recommendation and comments https
5. Any other relevant comments: please improve the discussion section as very few studies discussing the antibiotic susceptibility and other cases of infections with the same isolate.Please provide reason for not performing broth micro dilution ://doi.org/10.1099/acmi.0.000569.v1.5 © 2023 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Dear authors, I read the manuscript in details.Here are some recommendations for improving the manuscript: -Line 26: The bacteria in the genus Nocardia are filamentous.It recommends using the appropriate word because they are not true bacilli.-Line 96: Instead of " A 1% modified ZN… ", show how to do modified acid-fast staining correctly.Take into account that most Nocardia species are partially acid-fast when using modified acid-fast staining like Kinyoun method.-The history of medical interventions, including each immunomodulation, medicine, and any other patient's risk factors, should be provided in the case presentation section.
Is there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?YesReviewer 2